Parathyroid hormone (PTH) stimulates cAMP production via specific receptors in the hormone's target tissues, bone and kidney. We have recently described cell culture systems derived from bone and kidney containing PTH receptors coupled to formation of cAMP, in which regulation of the cellular response to the hormone can be investigated. Incubation with PTH desensitizes these target cells to a subsequent challenge by homologous hormone. In bone cells desensitization results, in large part, from a reduction in the number of cell surface PTH receptors, whereas the kidney cells lose very little PTH binding activity. Thus, the measurable fraction of available PTH binding sites, which are no longer coupled to cAMP production is greater in the kidney cell system relative to bone cell cultures. Our previous studies indicate that PTH is internalized by osteoblasts in calvariae from which the bone cell cultures are isolated and that this hormonal uptake occurs via receptor-mediated endocytosis. Such a process could account for cell surface receptor down-regulation. Resensitization of cells with decreased hormonal responsiveness might be effected by synthesis of new receptors, reinsertion of internalized receptors or reactivation of uncoupled receptors. We proposed to distinguish between these possibilities by determining the rate of recovery of desensitized cells incubated in PTH-free medium 1) when receptor synthesis is blocked, 2) when receptor uptake is inhibited, and 3) when receptor reinsertion is prevented. In this way, we will evaluate the role of a reactivation process in the maintenance of active cell surface receptor concentration by determining the capacity and velocity of such a process. We will attempt to accelerate recovery of available PTH receptors and PTH-stimulation of cAMP production by inhibiting changes in cell surface receptor concentration. We will then determine whether saturation of the reactivation mechanism shunts uncoupled receptors to an endocytotic pathway. Thus, we will test the idea that a branch point in the metabolism of PTH receptors occurs at the cell surface. In addition, we anticipate that these studies will lead to a better understanding of the maintenance of PTH responsiveness not only in desensitized cells but in naive cells as well. We expect that the mechanisms for regulation of active cell surface receptor concentration are different (at least quantitatively) only in these bone and kidney cell cultures and that we will be able to attribute these differences to new receptor synthesis, reinsertion of receptors and/or a process capable of reactivating receptors.